Anti-Inflammatory and Analgesic Activities of Flavonoid and Saponin Fractions from Zizyphus Lotus (L.) Lam

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South African Journal of Botany 74 (2008) 320–324 www.elsevier.com/locate/sajb

Anti-inflammatory and analgesic activities of flavonoid and saponin fractions from Zizyphus lotus (L.) Lam. ⁎ W. Borgi a, , M.-C. Recio b, J.L. Ríos b, N. Chouchane a

a Laboratory of Pharmacology, Faculty of Pharmacy, 5000 Monastir, Tunisia b Departament de Farmacologia, Facultat de Farmàcia, Universitat de Valencia, Av. Vicent Andrés Estellés s/n, 46100 Burjassot, Valencia, Spain Received 29 September 2007; received in revised form 12 January 2008; accepted 25 January 2008

Abstract

The effect of the flavonoid and saponin fractions from the leaves and root bark of Zizyphus lotus (200 mg/kg) was evaluated on carrageenan- induced paw edema in rats and on acetic acid-induced algesia in mice. In addition, two methanolic extracts from the plant (1 mg/ear) were tested on oxazolone-induced contact-delayed hypersensitivity (DTH) in mice. The effect of the different fractions was also evaluated in vitro on the nitrite production induced by lipopolysaccharide (LPS) in RAW 264.7 macrophages. The results showed that both the flavonoid and saponin fractions significantly inhibited paw edema, algesia, and nitrite production without affecting cell viability. Furthermore, the methanolic extracts of the leaves and root bark of Z. lotus were found to significantly inhibit the DTH induced by oxazolone, with the extract obtained from root bark being more active than that obtained from leaves. © 2008 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Analgesic activity; Anti-inflammatory activity; Delayed type hypersensitivity; Nitrite production; Zizyphus lotus

1. Introduction of acetic acid. We have also examined the effects of the extracts on nitrite production in RAW 264.7 macrophages. In addition, Many plants used in traditional medicine have the potential methanolic extracts from the root bark and leaves were tested on to provide pharmacologically active natural products. For oxazolone-induced contact-delayed type hypersensitivity example, several recent pharmacological studies (Nunes et al., (DTH) in mice. Finally, the cytotoxicity of Z. lotus fractions 1987; Adzu et al., 2001; Borgi et al., 2007) have focused on the on different cells was evaluated. various species of Zizyphus (Rhamnaceae), including Zizyphus lotus, a species commonly known as ‘sedra’ in Tunisia, where it 2. Materials and methods is used in traditional medicine as a pectoral demulcent to treat throat and broncho-pulmonic irritations (Le-Floc'h, 1983). In a 2.1. Reagents previous study, we reported on the anti-inflammatory and analgesic activities of Z. lotus root bark (Borgi et al., 2007). In Carrageenan (BDH Chemicals Ltd Poole England), and the present study, we have gone on to investigate the effects of acetylsalicylic acid (AAS) were from Adwya Laboratory, the flavonoid and saponin fractions of the root bark and leaves Tunisia. RAW 264.7 macrophages, lipopolysaccharide (LPS) ω of Z. lotus on both carrageenan-induced paw edema in rats and from Escherichia coli 0111:B4 (Sigma), N -nitro-L-arginine the algesia induced in mice by the intraperitoneal administration methyl ester (L-NAME) and DMEM medium all from Invitrogen, Langley, OK, USA. 96-well microplates, oxazolone, dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5- ⁎ Corresponding author. Tel.: +216 73 461000; fax: +216 73 461830. diphenyl tetrazolium bromide (MTT), Griess reagent and acetic E-mail address: [email protected] (W. Borgi). acid were purchased from Sigma.

2.2. Plant material allowed free access to drinking water. Animals fasted overnight before any experiments, all of which were conducted in accor- Z. lotus (L.) Lam. was collected in Cherahil-Monastir, Tunisia, dance with the guidelines established by the European Union on in 2005 and identified in the Botanic laboratory of the Faculty of Animal Care (CEE Council 86/609). Housing conditions and all Pharmacy in Monastir. A voucher specimen (No. 0269) has been in vivo experiments were approved and followed by the Insti- deposited in the Herbarium of the Department of Pharmacognosy, tutional Ethics Committee of the Faculty of Pharmacy (Univer- Faculty of Pharmacy, in Monastir, Tunisia. sity of Valencia, Spain).

2.3. Preparation of extracts 2.5. Anti-inflammatory activity

The root bark (RB) and leaves (L) were dried, coarsely 2.5.1. Carrageenan-induced rat paw edema powdered, and exhaustively extracted in a Soxhlet apparatus Wistar rats were divided into groups of six animals each. with methanol for 8 h to yield the methanolic extract (ME); ME- Edema was induced by injecting 0.05 ml of 1% carrageenan RB: 25.27% and ME-L: 15.28%. In order to obtain total oligo- subcutaneously into the sub-plantar region of the left hind paw mers flavonoids (TOF), the powdered root bark and leaves were (Winter et al., 1962). Flavonoid (TOF) and saponin (S) fractions macerated separately for 24 h in a mixture of acetone/water of the root bark (RB) and leaves (L) of Z. lotus (200 mg/kg) were (2:1). Each extract was then filtered and the acetone was eva- administered intraperitoneally (i.p.). The reference group received porated under low pressure. Tannins were removed from the acetylsalicylic acid (ASA, 300 mg/kg, i.p.) and the control group aqueous phase through precipitation with NaCl for 24 h at 5 °C received a solution of 9‰ NaCl (2.5 ml/kg, i.p). All drugs were and the supernatant was then extracted with ethyl acetate, con- administrated 15 min before the injection of carrageenan. The centrated, and precipitated with an excess of chloroform. The hind paw volume was measured with a plethysmometer (model precipitate containing TOF was separated by means of filtration 7150, Ugo Basile, Comerio, Italy) at 0 h (before carrageenan (Paris and Moyse, 1976a,b,c). The yield of TOF-RB and TOF-L injection) and then again at 0.5, 1, 2, 3, 4, 6, and 24 h. fractions was 0.12% and 0.13%, respectively. The presence of flavonoids was verified by the reaction of ‘cyanidine’ in the 2.5.2. Oxazolone-induced delayed type hypersensitivity in mice presence of hydrochloric acid and of magnesium. After release In accordance with the method previously described by Young of hydrogen, a coloring of orange with the red purple indicates and De Young (1989),femalemice(n= 6) were sensitized through the presence of the flavonoids. The revelation of the spots cor- topical application to the shaved ventral abdomen of 150 µl of a responding to the flavonoids on the plates of thin layer chro- 3% (w/v) solution of oxazolone in acetone on day 1. Challenge matography (TLC) is done by pulverization using a solution of was performed on day 6 by applying 10 µl of 1% oxazolone to AlCl3 in methanol 2%. The plates of TLC are then observed the inner and outer surfaces of both ears. Root bark (ME-RB) and under UV with 366 nm: the spots corresponding to the flavonoids leaf (ME-L) methanolic extracts (1 mg/ear) and dexamethasone have a yellow fluorescence at intense yellow (Paris and Moyse, (0.025 mg/ear) were applied topically (10 µl) to the inner and 1976a,b,c). outer surface of right ear on days 6, 7, and 8 after challenge. The The saponin fraction (S) was obtained as follows: powdered thickness of each ear was measured with a micrometer (Mitutoyo root bark and leaves were macerated separately in petroleum Series 293, Kawasaki, Japan) on days 7, 8, and 9 after challenge ether. After 24 h, each defatted plant material was macerated in just before drug application. The edema was calculated for each MeOH for another 24 h and then the filtrate was concentrated ear as the difference in thickness before treatment and 24, 48, and under low pressure. Ethyl ether was added to the methanolic 72 h after challenge. The activity was expressed as the inhibition extract and kept at −5 °C for 24 h. A precipitate was obtained percentage of ear thickness with reference to the control group. under low pressure and was separated by means of filtration (Boiteau et al., 1964). The yield of S-RB and S-L fractions was 2.6. Cytotoxicity assay 2.19% and 0.34%, respectively. The presence of saponins was verified as follow: after dissolution of the active ingredients in The cytotoxicity of Z. lotus extracts was determined with hot water, the tubes containing the solutions are agitated during the aid of the colorimetric assay (Mosmann, 1983). Cells were 15 s (2 agitations a second). The development of persistent foam exposed to the extracts (50–1000 µg/ml) in a microplate for which lasts more than 15 min indicates the presence of the 30 min (rat peritoneal neutrophils) or 1 day (RAW 264.7 saponins (Boiteau et al., 1964). macrophages) and then 100 µl/well of a 5 mg/ml solution of 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide 2.4. Animals (MTT) was added. The plates were then incubated at 37 °C until blue deposits were visible. The coloured metabolite was For studying the anti-inflammatory and analgesic activities, dissolved in dimethyl sulfoxide (100 µl per well). Absorbance adult Wistar rats (150–200 g) and Swiss albino mice (18–22 g) was measured at 490 nm with a Labsystems Muliskan MCC/ were obtained from the Pasteur Institute (Tunis, Tunisia). For 340 plate reader (Helsinki, Finland). Results are expressed as experiments on oxazolone-induced hypersensitivity in mice, absolute absorbance readings with a decrease indicating a animals were obtained from Harlan InterFauna Ibérica (Barce- diminution in cell viability. Controls received vehicle only and lona, Spain). All animals were fed a standard diet ad libitum and correspond to 100% viability. 322 W. Borgi et al. / South African Journal of Botany 74 (2008) 320–324

Table 1 Effect of Z. lotus extracts on carrageenan-induced paw edema Group Dose Paw edema volume mean (10− 2 ml)±s.e.m. (%I) (n=6) (mg/kg) 0.5h1h2h3h4h6h24h Control 1 22.37±1.41 38.87±3.29 68.00±6.31 96.75±8.87 106.25±10.04 114.75±11.43 77.25±6.77 ASA 300 12.25±0.62*** 14.50±1.55*** 17.25±1.31*** 27.25±4.26*** 31.00±3.18*** 42.50±3.77** 46.25±6.86* (45) (63) (75) (72) (71) (63) (40) Control 2 28.25±2.50 63.25±4.99 112.50±9.87 157.25±12.19 170.50±14.14 182.00±14.21 110.00±13.70 TOF-RB 200 15.66±6.67** 24.33±3.84*** 33.00±3.78*** 39.00±3.60*** 44.00±2.30*** 57.33±6.17*** 45.33±5.78** (45) (62) (71) (75) (74) (68) (59) TOF-L 200 23.66±2.90ns 36.33±4.84* 69.33±5.48* 84.00±10.69** 91.66±13.38* 112.33±17.36* 72.66±7.31ns (16) (43) (38) (47) (46) (38) (34) Control 3 24.00±2.12 35.20±5.32 66.60±1252 120.20±13.76 151.80±18.21 147.60±11.96 92.80±3.80 S-RB 200 11.66±1.33** 11.00±2.00** 10.66±0.88** 12.00±1.15** 11.33±0.33** 15.66±2.33** 21.66±5.92** (51) (69) (84) (90) (93) (89) (77) S-L 200 17.33±0.33ns 22.00±1.00ns 24.33±1.33* 42.33±1.66** 57.33±8.00** 56.66±9.20** 51.66±11.20** (28) (38) (64) (65) (62) (62) (44) Data are expressed as mean±s.e.m. (n=6), *Pb0.05, **Pb0.01, ***Pb0.001, ns: not significant vs. control. Statistical analysis was performed with Student's t-test. %I: percentage inhibition, TOF-RB: total oligomers flavonoids of root bark, TOF-L: total oligomers flavonoids of leaves, S-RB: saponins of root bark, S-L: saponins of leaves, ASA: acetylsalicylic acid.

2.7. Inhibition of nitrite production in RAW 264.7 macrophages 30 min was quantified. A significant reduction in writhing as compared to the control group was considered to be an anti- RAW 264.7 macrophages were cultured in a DMEM medium nociceptive response (Koster et al., 1959). containing 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 10% foetal bovine serum. Cells were removed 2.9. Data analysis from the tissue culture flask with a cell scraper and resuspended until a final relation of 1×106 cells/ml was reached. The cells Data are presented as the mean±standard error (s.e.m.). Statis- were then co-incubated in a 96-well culture plate (200 µl) with tical analysis was performed using Student's t-test. The signifi- 1 µg/ml of lipopolysaccharide (LPS) at 37 °C for 24 h in the cance of difference was considered to include values of Pb0.05. presence of extracts (50 and 100 µg/ml), DMSO (control), or Nω- nitro-L-arginine methyl ester (L-NAME) as a reference drug. The 3. Results and discussion supernatants were used directly to measure nitrite production by first mixing them with Griess reagent and then reading the ab- In a previous study (Borgi et al., 2007), we reported on the anti- sorbance at 570 nm in a microplate reader (Bas et al., 2007). inflammatory and analgesic properties of the aqueous and metha- Nitrite production was assessed as the index of NOS activity. nolic extracts (200 mg/kg, i.p.) of the root bark of Z. lotus.The results showed that the polar extracts (aqueous and methanolic 2.8. Antinociceptive activity extracts) were more active than the chloroformic and the ethyl acetate extracts. We also verified the presence of flavonoids Swiss mice were selected 1 day prior to the assay and were and saponins in these extracts, so we direct our research towards divided into different groups of six animals each. The control the effect of these fractions and their participation in the anti- group was treated with 10 mg/kg, i.p. of 9‰ NaCl. The positive inflammatory activity. The results obtained in this study indicate control (reference) group received acetylsalicylic acid (ASA, that these fractions at the dose of 200 mg/kg administered 200 mg/kg, i.p.) while the remaining groups were given TOF-RB, intraperitoneally present a significant effect against carrageenan- TOF-L, S-RB, and S-L of Z. lotus (200 mg/kg, i.p.). Fifteen induced inflammation and their effect was more important. minutes later, 1% acetic acid (10 ml/kg. i.p.) was injected into In carrageenan-induced rat paw edema (Table 1), the extracts each animal and the amount of writhing during the following TOF-RB, TOF-L, S-RB, and S-L (all at 200 mg/kg, i.p.)produced

Table 2 Inhibitory effects of Z. lotus extracts on oxazolone-induced contact-delayed hypersensitivity ear edema in mice Treatment ΔT (µm) %I 24 h 48 h 72 h 24 h 48 h 72 h Control 256.73±32.28 166.27±13.05 148.13±17.90 ––– ME-L 132.26±13.04** 129.00±14.07ns 95.69±12.26* 48 22 35 ME-RB 108.30±10.27*** 106.86±13.05*** 78.90±7.89** 58 36 47 Dexamethasone 65.29±6.89*** 51.23±8.55*** 28.52±15.38*** 75 69 81 Z. lotus extracts dose=1 mg/ear×3 applications, Dexamethasone 0.05 mg/ear, ΔT=increase in ear thickness, %I=inhibition percentage. Data are expressed as mean± s.e.m. (n=6), *Pb0.05, **Pb0.01, ***Pb0.001, ns: not significant vs. control. Statistical analysis was performed with Student's t-test. (ME-L: methanolic extract of leaves; ME-RB: methanolic extract of root bark). W. Borgi et al. / South African Journal of Botany 74 (2008) 320–324 323 a significant reduction of the edema throughout the entire period Table 4 of observation. Interestingly, the reduction of the edema by 75% Effect of Z. lotus fractions on acetic acid-induced writhing in mice and 90% after administration of TOF-RB and S-RB, respectively, Group (n=6) Dose Number of writhes Inhibition of writhing was highest at 3 h, whereas at the same time, TOF-L and S-L (mg/kg) (per 30 min) response (%) inhibited the edema by 47% and 65%, respectively. At any rate, Control 1 – 100.84±5.64 – these results indicate that the anti-inflammatory effect may be ASA 200 36.95±6.27*** 63 – – related, at least in part, to the presence of flavonoids and saponins. Control 2 102.40±6.71 TOF-RB 200 19.00±0.00*** 81 There have been previous reports on the anti-inflammatory acti- TOF-L 200 74.33±9.05* 27 vity of these types of compounds, including several on their S-RB 200 54.33±16.41* 47 possible mechanism of action. In general, flavonoids act as po- S-L 200 12.00±2.68*** 88 tential inhibitors of cyclooxygenase, lipoxygenase, and nitric Values are expressed as mean±s.e.m. *Pb0.05, ***Pb0.001. Statistical ana- oxide synthase as well as being antioxidants (Rao et al., 2005; lysis was performed with Student's t-test. (TOF-RB: total oligomers flavonoids Rotelli et al., 2003; Havsteen, 2002; Middleton and Kandaswami, of root bark, TOF-L: total oligomers flavonoids of leaves, S-RB: saponins of 1992; Middleton et al., 2000; Di Carlo et al., 1999). In contrast, root bark, S-L: saponins of leaves, ASA: acetylsalicylic acid). saponin mechanisms are related to those of steroids, the release of different enzymes, and the arachidonic acid metabolism (Ríos extracts were not toxic for cells with viability greater than 95%. et al., 2000; Safayhi and Sailer, 1997). The cytotoxicity was observed at high concentration tested (500 In the DTH test, examination of challenged ears showed that and 1000 µg/ml) with viability percentage 90%. Saponins and oxazolone induced a pronounced inflammatory reaction com- especially the methanolic extract of the leaves (ME-L) signifi- pared to ears that had not been challenged. Of the extracts as- cantly inhibited nitrite production by 50% and 74%, respectively, sayed, the activity of the ME-RB extract was higher than that of while the TOF-RB and ME-RB extracts of the root bark only had ME-L, although both extracts significantly reduced ear swelling amoderateeffectat100μg/ml, whereas at 50 μg/ml both extract (Table 2). Indeed, the ME-RB extract reduced the edema in all induced the nitrite production, especially S-RB, which would be phases of the process by 58%, 36%, and 47% at 24, 48, and 72 h of interest for a future research. These results are in contrast with after challenge, respectively, whereas ME-L only reduced the the in vivo test, in which the extracts obtained from RB were edema by 48% and 35% at 24 and 72 h, respectively. It is worth clearly more active, a fact which suggests that the inhibitors of noting that in this test, only corticosteroids are active because nitric oxide synthase are different from the substances responsible specific inhibitors of the arachidonic acid metabolism have no for the anti-inflammatory activity. effect on the inflammatory reaction (Cavey et al., 1990; Zunic In acetic acid-induced writhing in mice, the TOF-RB and S- et al., 1998; Meurer et al., 1988). These findings suggest that the L fractions (each at 200 mg/kg) reduced the writhing by 81 and mechanism of action of Z. lotus saponins may be related to a 88%, respectively, whereas TOF-L and S-RB only inhibited the corticosteroid mechanism. writhing by 27% and 47%, respectively (Table 4). This type of NO plays an important role in diverse physiological processes writhing in mice has been associated with the liberation of including the immune response and inflammation (Ashok et al., endogenous substances including serotonin, histamine, prosta- 1999). For this reason, numerous plants have been investigated as glandin, and bradykinin (Collier et al., 1968). The results inhibitors of NO production in inflammatory reactions (Lee et al., obtained in this test thus suggest that while Z. lotus extracts 2005; Escandell et al., 2006; Choi et al., 2007). In order to test the possess peripheral analgesic properties, this particular activity is anti-inflammatory activity of Z. lotus extracts, we investigated probably unrelated to their anti-inflammatory properties. their effects on LPS-induced nitrite production in RAW 264.7 macrophages (Table 3). We first tested the cell viability in the 4. Conclusion presence of the test extract to determine the toxicity against murine RAW 264.7 macrophages or rat peritoneal neutrophils In summary, our results have demonstrated that Z. lotus (PMNL). The results showed that at 50 and 100 µg/ml, Z. lotus exhibits anti-inflammatory activity, inhibits NO production, and reduces peripheral algesia. These results confirm the potential Table 3 interest in Z. lotus as a medicinal plant and open the way for Effects of Z. lotus extracts on nitrite production in RAW 264.7 murine future research focussing on the active extracts or fractions. Of 6 macrophages (1×10 cells/ml) special interest are the S-RB fraction as a potential source of anti- Product TOF-L S-L ME-L TOF-RB S-RB ME-RB L-NAME inflammatory principles, the ME-RB extract as a possible source 50 µg/ml (−23) 15 30 (−3) (−67) (−8) – of anti-inflammatory agents in DTH reactions, and the TOF-RB 100 µg/ml 7 50 74 37 14 21 – and S-L fractions as potential sources of analgesic drugs. 1000 µM –––– –– 105 Nitrite production is assessed as the index of nitric oxide synthase activity. References Values are expressed as inhibition of nitrite production. (TOF-RB: total oligomers flavonoids of root bark, TOF-L: total oligomers flavonoids of leaves, Adzu, B., Amos, S., Wambebe, C., Gamaniel, K., 2001. Antinociceptive activity S-RB: saponins of root bark, S-L: saponins of leaves, ME-L: methanolic extract of Zizyphusspina-christi root bark extract. Fitoterapia 72, 344–350. ω of leaves; ME-RB: methanolic extract of root bark; L-NAME: N -nitro-L- Ashok, R.A., Mukundan, A., Steven, B.A., 1999. Nitric oxide synthase and arginine methyl ester was used as reference). Notice: The negative values mean cyclooxygenases: distribution, regulation, and intervention in arthritis. Cur- an increase of nitrite production. rent Opinion in Rheumatology 11, 202–209. 324 W. Borgi et al. / South African Journal of Botany 74 (2008) 320–324

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